Strain-Specific Gifsy-1 Prophage Genes Are Determinants for Expression of the RNA Repair Operon during the SOS Response in Salmonella enterica Serovar Typhimurium

ABSTRACT The adaptation of Salmonella enterica serovar Typhimurium to stress conditions involves expression of genes within the regulon of the alternative sigma factor RpoN (σ54). RpoN-dependent transcription requires an activated bacterial enhancer binding protein (bEBP) that hydrolyzes ATP to remodel the RpoN-holoenzyme-promoter complex for transcription initiation. The bEBP RtcR in S. Typhimurium strain 14028s is activated by genotoxic stress to direct RpoN-dependent expression of the RNA repair operon rsr-yrlBA-rtcBA. The molecular signal for RtcR activation is an oligoribonucleotide with a 3′-terminal 2′,3′-cyclic phosphate. We show in S. Typhimurium 14028s that the molecular signal is not a direct product of nucleic acid damage, but signal generation is dependent on a RecA-controlled SOS-response pathway, specifically, induction of prophage Gifsy-1. A genome-wide mutant screen and utilization of Gifsy prophage-cured strains indicated that the nucleoid-associated protein Fis and the Gifsy-1 prophage significantly impact RtcR activation. Directed-deletion analysis and genetic mapping by transduction demonstrated that a three-gene region (STM14_3218-3220) in Gifsy-1, which is variable between S. Typhimurium strains, is required for RtcR activation in strain 14028s and that the absence of STM14_3218-3220 in the Gifsy-1 prophages of S. Typhimurium strains LT2 and 4/74, which renders these strains unable to activate RtcR during genotoxic stress, can be rescued by complementation in cis by the region encompassing STM14_3218-3220. Thus, even though RtcR and the RNA repair operon are highly conserved in Salmonella enterica serovars, RtcR-dependent expression of the RNA repair operon in S. Typhimurium is controlled by a variable region of a prophage present in only some strains. IMPORTANCE The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria. In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon. This work identifies key elements of a RecA-dependent pathway that generates the signal for RtcR activation in strain 14028s. This signaling pathway requires the presence of a specific region within the prophage Gifsy-1, yet this region is absent in most other wild-type Salmonella strains. Thus, we show that the activity of a widely conserved regulatory protein can be controlled by prophages with narrow phylogenetic distributions. This work highlights an underappreciated phenomenon where bacterial physiological functions are altered due to genetic rearrangement of prophages.

Plasmid construction. PCR amplification for plasmid construction used OneTaq DNA polymerase or Q5 High-Fidelity DNA polymerase, and ligation reactions used T4 DNA ligase, according the manufacturers recommended protocols (NEB, Ipswich, MA). Restriction enzymes were purchased from NEB. Plasmid products were transformed into chemically competent DH5α; for use in Salmonella, plasmids were replicated in the restrictionmodification + LT2 strain MS1868 prior to transformation into relevant strains.
Plasmid pJK14 was constructed by PCR amplifying the 14028s recA gene, with the addition of flanking NdeI and XhoI cut sites (oligos 51 & 54). The amplicon was digested with NdeI and XhoI, and the NdeI-XhoI fragment was introduced into the expression vector pSRK-Tc (9). pJK15 was constructed similarly, except that overlap extension PCR (10) was first used to introduce the single base pair mutation that converts the Glu 39 GAA codon to a Lys AAA codon. The recA gene was amplified from 14028s gDNA in two pieces using oligos 51 & 52 and 53 & 54); oligos 52 and 53 are direct complements that introduce the single G → A mismatch. The two fragments were then used in a subsequent PCR reaction, primed by their overlapping ends to generate the complete recA730 allele flanked by NdeI and XhoI for introduction into pSRK-Tc (11).
For pCH6, a PCR product encoding a truncated copy of S. Typhimurium RtcR beginning at Leu 179 was generated with the addition of flanking NdeI and XhoI cut sites (oligos 55 & 56) and TOPO cloned into pCR2.1 to make pCH4; the NdeI-XhoI fragment from pCH4 was then introduced into pSRK-Tc. Plasmid constructs were confirmed by Sanger sequencing (oligos 57 & 58) (Genewiz, South Plainfield, NJ).
To construct pJK19, a single-copy reporter plasmid in which rsrp is fused to lacZY, the regulatory region encompassing the transcription start site (TSS) of rsr through 634 bp of the N-terminal domainencoding region of rtcR was PCR amplified from 14028s genomic DNA with the addition of NotI and HindIII cut sites (oligos 59 & 60). The NotI-HindIII con promoter region of pAG620 (12) was replaced with the NotI-HindIII rsrp fragment; ligations were additionally digested with KpnI, which cuts within the con promoter region to prevent re-ligation of pAG602. Appropriate expression of LacZ was confirmed by plating S. Typhimurium WT, ΔrtcR, and ΔrpoN strains expressing RecA730 on X-Gal-containing medium. pJK21 was generated by PCR amplifying the STM14_3218-3220 putative operon sequence from 14028s genomic DNA, with the addition of flanking NheI and HindIII cut sites (oligos 61 & 62). Amplicons were digested with NheI and HindIII, and the fragment was introduced into pBAD30 (13). The resulting plasmid was confirmed by Sanger sequencing at Genewiz (South Plainfield, NJ) using standard pBAD sequencing primers and full-length plasmid sequencing at Plasmidsaurus (Eugene, OR). Plasmids pAK101, pAK102 and pAK103 were constructed by PCR amplifying 14028s STM14_3218, STM14_3218-3220, and STM14_3219-3220 with the addition of flanking NdeI and XhoI cut sites (oligos 99 & 100, 99 & 101, 102 & 101, respectively). The amplicons were digested with NdeI and XhoI, and the NdeI-XhoI fragments were introduced into pSRK-Tc (9). The resulting plasmids were confirmed by full-length plasmid sequencing at Plasmidsaurus (Eugene, OR).
For the construction of pP rsr -lacZ, a fragment containing 373 bp of the rtcR-rsr intergenic sequence and part of rtcR was generated by PCR amplification from S. Typhimurium LT2 genomic DNA with the addition of flanking XbaI and BamHI restriction sites (oligos 63 &64). The amplicon was inserted into pGEM-T (Promega); the resulting plasmid was digested with XbaI and BamHI and the XbaI-BamHI fragment containing the rsr promoter was isolated by agarose gel extraction and introduced into the MCS of p16R (pACYC184 with the BamHI to SalI region replaced by the E. coli lacZ gene; provided by Timothy Hoover). Tables-Table S1. Bacterial and prophage genes identified in Tn5 mutagenesis screen.   Data shown is representative of at least 2 biological replicates; error bars represent ±1 standard deviation. Significant differences in beta-galactosidase activity between uninduced and induced samples are indicated by asterisks above horizontal bars centered between the compared data sets; significance differences in beta-galactosidase activity for the RecA730 expression strain (uninduced or induced) in pairwise comparison to the RecA expression strain (uninduced or induced) are indicated by asterisks above the RecA730 samples (**, P value < 0.01).  Figure S2. RecA is not required for a constitutively active RtcR variant to stimulate RpoN-dependent transcription from rsrp. A XylE assay was performed to assess transcription from rsrp in the ΔrecA::kan Δrsr::xylE reporter strain (JEK26) carrying pCH6 which expresses a constitutively active variant of RtcR (RtcRcon). RtcRcon lacks the regulatory domain and is not dependent on sensing a signal for activation (1). Cells were assayed after growth in the presence of 0.2% glucose to repress expression of the plasmid or 50 μM IPTG to induce expression (90 min). Data shown is representative of 3 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Statistical significance was determined by Student's t test (*P < 0.001). Figure S3. RtcR is not activated to stimulate RpoN-dependent transcription of the E. coli rtcBA operon during the SOS response. (A) Crystal violet staining and bright field microscopy (1000x magnification) of E. coli cells carrying plasmids that express S. Typhimurium WT RecA (pJK14) or RecA730 (pJK15), grown in the presence of 0.2% glucose to repress expression from lacp or 1 mM IPTG to induce expression (90 min). (B) XylE activity assays were performed to assess activation of the rtcBp promoter in the E. coli reporter strain (ACW2) carrying pJK14 or pJK15. Mid-log phase cultures were split and one of each pair had 1 mM IPTG added to induce RecA or RecA730 expression from pJK14 or pJK15, respectively, while the other was uninduced. After 90 min growth the cultures were assayed for XylE activity. ACW2 carrying pCH6, which expresses the constitutively-active RtcRcon, was utilized as a positive control; XylE activity following treatment with 50 M IPTG for 90 min was 8.0  0.3 nmol/min/10 8 cells (234-fold higher than uninduced); data not included in bar graph. Data shown is representative of 3 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Statistical significance for induced versus uninduced, as determined by Student's t test, was P > 0.05 (not significant).
B. XylE Activity (nmol/min/10 8 cells) Figure S4. Reporter assays to assess activation of transcription from rsrp in deletion mutants for genes that were identified in the Tn5 mutagenesis screen. (A) Beta-galactosidase assays were conducted for single gene deletion mutants co-maintaining pJK15 (RecA730 expression vector) and pJK19 (rsrp-lacZ reporter); the indicated gene (on x-axis) was replaced with a kan resistance marker. (B) XylE activity assays were conducted on Δrsr::xylE reporter strains with select single gene deletion mutants (genes indicated on x-axis) and carrying pJK15. For (A) and (B), cultures were split at mid-log and assayed after 90 min growth without inducing agent or with 1 mM IPTG. All data shown is representative of at least 3 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Significant differences in XylE or beta-galactosidase activity between uninduced and induced (RecA730 expression) samples are indicated by asterisks above horizontal bars centered between the paired samples; significance differences in XylE or beta-galactosidase activity for mutant strains and treatments (uninduced or induced) in comparison to the WT counterpart are indicated by asterisks above the mutant samples (*, P value < 0.05; **, P value < 0.01).

A.
B.  Figure S5. RtcR activation shows a dependence on growth phase. XylE assays were performed with the 14028s WT Δrsr::xylE reporter strain containing pJK15 (RecA730 expression vector). Overnight cultures for each biological replicate were subcultured 1:100 in fresh LB-Tet and aliquots were taken for induction with 1 mM IPTG at the transition from lag to logarithmic growth (0.15 OD600), early-to mid-log phase (0.3 and 0.4 OD 600 ), and late-log phase (0.6 and 0.9 OD 600 ). The uninduced samples were taken at 0.4 OD 600 . Induced and uninduced samples were grown for an additional 90 min and cells were harvested to determine XylE activity. Activity increased for samples induced for RecA730 expression from late lag to early-to mid-log phases and dramatically decreased for samples induced during late-log growth. Data shown is for at least 3 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Significant differences in XylE activity between uninduced and induced samples are indicated by asterisks above the horizontal bar centered between the paired samples; significance differences in XylE activity for induced samples in comparison to the peak activity (0.4 OD600 samples) is indicated by asterisks above the samples (*, P value < 0.05, **, P value < 0.01). XylE assays were performed with the 14028s WT and STM14_3218-3220 Δrsr::xylE reporter strains, which contain no plasmids. Cultures were split at mid-log phase and one of each pair was treated with 3 M MMC to initiate the SOS response for RtcR activation conditions; cells were assayed after 90 min growth. Data shown is for 4 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Significant differences in XylE activity between uninduced and induced samples are indicated by asterisks above the horizontal bar centered between the paired samples; significance differences in XylE activity for the mutant strain and treatments (uninduced or induced) in comparison to the 14028s WT counterpart is indicated by asterisks above the mutant samples (*, P value < 0.05, **, P value < 0.01).  Table S3). Expression from lacp was induced with 1 mM IPTG. The RecA730 expression vector cannot be co-maintained with these pSRK-Tc-based plasmids, so treatment with 3 M MMC was utilized to induce RtcR activation conditions. Data shown is for at least 3 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Significant differences in XylE activity between uninduced and induced samples are indicated by asterisks above the horizontal bar centered between the paired samples; significant differences in XylE activity in comparison to the 14028s WT counterpart (first condition in (A) and (B)) is indicated by asterisks above the samples (*, P value < 0.05, **, P value < 0.01).    (6). The supernatants from uninduced samples for WT, STM14_3218-3220, and rtcR strains had equivalent infective phage particle concentrations for Gifsy-1 (8.7E+03, 6.4E+03, and 5.5E+03, respectively) and for all 3 Gifsy phages (1.4E+05, 1.6E+05, and 1.4E+05, respectively). The infective phage particle concentrations for supernatants from MMC-induced cultures are shown in the bar graphs. Data shown is for 6 biological replicates, each with two technical replicates; error bars represent ±1 standard deviation. Significant differences for phage titer in comparison to the WT is indicated by asterisks above the mutant samples (*, P value < 0.05, **, P value < 0.01).